A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.
Dementia
Conditions of dementia are frequently characterised by a progressive accumulation of intracellular and/or extracellular deposits of proteinaceous structures such as β-amyloid plaques and neurofibrillary tangles (NFTs) in the brains of affected patients. The appearance of these lesions largely correlates with pathological neurofibrillary degeneration and brain atrophy, as well as with cognitive impairment (see, e.g., Mukaetova-Ladinska et al., 2000). In Alzheimer's disease, both neuritic plaques and NFTs contain paired helical filaments (PHFs), of which a major constituent is the microtubule-associated protein tau (see, e.g., Wischik et al., 1988a). Plaques also contain extracellular β-amyloid fibrils derived from the abnormal processing of amyloid precursor protein (APP) (see, e.g., Kang et al., 1987). An article (Wischik et al., 2001) discusses in detail the putative role of tau protein in the pathogenesis of neurodegenerative dementias. Loss of the normal form of tau, accumulation of pathological PHFs, and loss of synapses in the mid-frontal cortex all correlate with associated cognitive impairment. Furthermore, loss of synapses and loss of pyramidal cells both correlate with morphometric measures of tau-reactive neurofibrillary pathology, which parallels, at a molecular level, an almost total redistribution of the tau protein pool from a soluble to a polymerised form (i.e., PHFs) in Alzheimer's disease (see, e.g., Mukaetova-Ladinska et al., 1993).
Tau exists in alternatively-spliced isoforms, which contain three or four copies of a repeat sequence corresponding to the microtubule-binding domain (see, e.g., Goedert et al., 1989a; Goedert et al., 1989b). Tau in PHFs is proteolytically processed to a core domain (see, e.g., Wischik et al., 1988a; Wischik et al., 1988b; Novak et al., 1993) which is composed of a phase-shifted version of the repeat domain; only three repeats are involved in the stable tau-tau interaction (see, e.g., Jakes et al., 1991). Once formed, PHF-like tau aggregates act as seeds for the further capture and provide a template for proteolytic processing of full-length tau protein (see, e.g., Wischik et al., 1996a).
The phase shift which is observed in the repeat domain of tau incorporated into PHFs suggests that the repeat domain undergoes an induced conformational change during incorporation into the filament. During the onset of AD, it is envisaged that this conformational change could be initiated by the binding of tau to a pathological substrate, such as damaged or mutated membrane proteins (see, e.g., Wischik et al., 1997).
In the course of their formation and accumulation, PHFs first assemble to form amorphous aggregates within the cytoplasm, probably from early tau oligomers which become truncated prior to, or in the course of, PHF assembly (see, e.g., Mena et al., 1995; Mena et al., 1996). These filaments then go on to form classical intracellular NFTs. In this state, the PHFs consist of a core of truncated tau and a fuzzy outer coat containing full-length tau (see, e.g., Wischik et al., 1996a). The assembly process is exponential, consuming the cellular pool of normal functional tau and inducing new tau synthesis to make up the deficit (see, e.g., Lai et al., 1995). Eventually, functional impairment of the neurone progresses to the point of cell death, leaving behind an extracellular NFT. Cell death is highly correlated with the number of extracellular NFTs (see, e.g., Wischik et al., 2001). As tangles are extruded into the extracellular space, there is progressive loss of the fuzzy outer coat of the PHFs with corresponding loss of N-terminal tau immunoreactivity, but preservation of tau immunoreactivity associated with the PHF core (see, e.g., Bondareff et al., 1994).
Methylthioninium Chloride (MTC)
Methythioninium Chloride (MTC) (also known as Methylene blue (MB); methylthionine chloride; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathionium chloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet; and Urolene Blue®) is a low molecular weight (319.86), water soluble, tricyclic organic compound of the following formula:

MTC is a well known phenothiazine dye and redox indicator and has also been used as an optical probe of biophysical systems, as an intercalator in nanoporous materials, as a redox mediator, and in photoelectrochromic imaging.
MTC and other diaminophenothiazines have been described as inhibitors of protein aggregation in diseases in which proteins aggregate pathologically.
In particular, diaminophenothiazines including MTC have been shown to inhibit tau protein aggregation and to disrupt the structure of PHFs, and reverse the proteolytic stability of the PHF core (see, e.g., Wischik et al., 1996b). Such compounds were disclosed for use in the treatment or prophylaxis of various diseases, including Alzheimer's disease.
Wischik et al., 2007a discloses certain specific diaminophenothiazine compounds related to MTC which are useful as drugs, for example in the treatment of Alzheimer's disease.
Additionally, Schweiger et al., 2005, discusses radiolabelled phenothiazines, and their use in diagnosis and therapy, for example, of tauopathies.
MTC has also been disclosed for other medical uses. For example it is currently used to treat methemoglobinemia (a condition that occurs when the blood cannot deliver oxygen where it is needed in the body). MTC is also used as a medical dye (for example, to stain certain parts of the body before or during surgery); a diagnostic (for example, as an indicator dye to detect certain compounds present in urine); a mild urinary antiseptic; a stimulant to mucous surfaces; a treatment and preventative for kidney stones; and in the diagnosis and treatment of melanoma.
MTC has been used to treat malaria, either singly (see, e.g., Guttmann and Ehrlich, 1891) or in combination with chloroquine (see, e.g., Schirmer et al., 2003; Rengelshausen et al., 2004).
MTC (under the name Virostat®, from Bioenvision Inc., New York) has also shown potent viricidal activity in vitro. Specifically Virostat® is effective against viruses such as HIV and West Nile Virus in laboratory tests. Virostat® is also currently in clinical trials for the treatment of chronic Hepatitis C, a viral infection of the liver. The virus, HCV, is a major cause of acute hepatitis and chronic liver disease, including cirrhosis and liver cancer.
MTC, when combined with light, can also prevent the replication of nucleic acid (DNA or RNA). Plasma, platelets and red blood cells do not contain nuclear DNA or RNA. When MTC is introduced into the blood components, it crosses bacterial cell walls or viral membrane then moves into the interior of the nucleic acid structure. When activated with light, the compound then binds to the nucleic acid of the viral or bacterial pathogen, preventing replication of the DNA or RNA. Because MTC can inactivate pathogens, it has the potential to reduce the risk of transmission of pathogens that would remain undetected by testing.
Oral and parenteral formulations of MTC have been commercially available in the United States, usually under the name Urolene Blue®.
Leuco Methylthioninium (LMT)
MTC, a phenothiazin-5-ium salt, may be considered to be an “oxidized form” in relation to the corresponding 10H-phenothiazine compound, N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine (“Leuco methylthionine”, LMT), which may be considered to be a “reduced form”:

The “reduced form” (or “leuco form”), LMT, is known to be unstable and can be readily and rapidly oxidized to give the corresponding “oxidized” form, e.g., MTC.
It has been shown that human erythrocytes sequentially reduce and take up MTC; that MTC itself is not taken up by the cells; that it is the reduced form of MTC that crosses the cell membrane; that the rate of uptake is enzyme dependent; and that both MTC and reduced MTC are concentrated in cells (LMT, once inside the cell oxidises to MT+ and an equilibrium is established). See, e.g., May et al., 2004.
MTC and similar drugs are taken up in the gut and enter the bloodstream. Unabsorbed drug percolates down the alimentary canal, to the distal gut. One important undesired side-effect is the effect of the unabsorbed drug in the distal gut, for example, sensitisation of the distal gut and/or antimicrobial effects of the unabsorbed drug on flora in the distal gut, both leading to diarrhoea. Therefore, it is desirable to minimize the amount of drug that percolates to the distal gut. By increasing the drug's uptake in the gut (i.e., by increasing the drug's bioavailability), dosage may be reduced, and the undesired side-effects, such as diarrhoea, may be ameliorated. Since it is the reduced form of MTC that is taken up by cells, it may be desirable to administer the reduced form to patients. This may also reduce reliance on the rate limiting step of enzymatic reduction.
Wischik et al., 2002 describes the use of reduced forms of certain diaminophenothiazines for the treatment of protein aggregating diseases, primarily tauopathies.
Wischik et al., 2007b describes certain 10H-phenothiazine-3,7-diaminium salts, effective as drugs or pro-drugs for the treatment of diseases including Alzheimer's disease. These compounds are also in the “reduced” or “leuco” form when considered in respect of MTC. Among the examples described therein are the di-HCl salt (LMT.2HCl), the di-HBr salt (LMT.2HBr), and the di-HI salt (LMT.2HI).
Wischik et al., 2012 describe further 10H-phenothiazine-3,7-diaminium salts, including certain sulfonate salts, effective as drugs or pro-drugs for the treatment of diseases including Alzheimer's disease. Among the examples described therein are the di-mesylate salt (LMT.2MsOH; LMTM), the di-edisylate salt (LMT.2EsOH), the di-tosylate salt (LMT.2TsOH), the di-benzenesulfonate salt (LMT.2BSA), the ethanedisulfonate salt (LMT.EDSA), the propanedisulfonate salt (LMT.PDSA), and the naphth-1,7-di-sulfonate salt (LMT.NDSA).
Galey et al., 2010, describes certain 10H-phenothiazine-2,8-diamine compounds of the following formula which allegedly have biocidal activity and are useful in the agro-food industry and in the treatment of effluent. The document describes methods for preparing the 10H-phenothiazine-2,8-diamine compounds using a step of cross-coupling of anilines and halo benzenes followed by sulphur insertion and phenothiazine ring formation. According to the general teaching provided therein, the substituents on the pendant amino groups (i.e., —R2a, —R2b, —R8a, —R8b) may be present throughout synthesis (i.e., may be present before cross-coupling and phenothiazine ring formation) or may be added later, after phenothiazine ring formation, by alkylation, reductive amination, or acylation. However, in all of the worked examples, only the first method was used; that is, the substituents on the pendant amino groups, if present, were already attached before the cross-coupling reaction was carried out. Despite the lack of worked examples, and without any supporting evidence, the authors appear to allege that the proposed addition of substituents on the pendant amino groups after phenothiazine ring formation, according to the second method, would be selective for the pendant amino groups over the ring nitrogen that forms part of the phenothiazine ring (see, e.g., paragraph [0182] therein).

Booth et al., 2001, describes methods of preparing a range of tricyclic compounds of the following formula which allegedly have anti-viral activity. A small number of compounds were prepared using “singleton synthesis” and characterised; however, none of these compounds is a phenothiazine. See, e.g., pages 98-101 therein. A large number of compounds were prepared using “combinatorial chemistry synthesis” by reductive amination of a suitable amine using an aldehyde/ketone and sodium triacetoxyborohydride. See, e.g., pages 10-15 therein. The combinatorial products were characterised by mass spectrometry only, and chemical structures were tentatively assigned accordingly, without further supporting evidence. No yields were reported. Of the 458 compounds listed on pages 21-35 therein and the 446 compounds shown in the table at pages 36-98 therein, only 20 compounds are phenothiazines (i.e., Examples 88, 89, 324, and 415-431). However, each one is a 10H-phenothiazine-2-amine, and not a 10H-phenothiazine-3,7-diamine.
Improved Methods of Synthesis
It is generally desirable that chemical compounds which are intended to be used as pharmaceuticals are provided in a form that is sufficiently free of undesired impurities. This is especially true for chemical compounds that are intended to be used as part of long-term therapy, for example, daily administration for a period of months or years (or, indeed, indefinitely).
The presence of even relatively small amounts of certain undesirable impurities can render a chemical compound unacceptable for use in therapy, for example, accordingly the specifications set by national regulatory bodies (e.g., the US Food and Drug Administration, the European Medicines Agency, etc.).
Among the many undesired impurities are certain metals, including especially chromium (Cr). It is often extremely difficult to remove these metal impurities from a chemical compound that has been prepared by a method of chemical synthesis which used them.
For example, a method of chemical synthesis which employs, as an oxidizing agent, a chromium compound (e.g., chromate, CrO42−; dichromate, Cr2O72−) often yields a product with residual chromium, which cannot easily (or at all) be reduced to acceptable levels.
As discussed above, thioninium salts (such as MTC), thionines (such as LMT), and thionine di-salts (such as LMT.2EsOH) have utility in the long-term treatment of chronic conditions (such as Alzheimer's disease) and accordingly must be provided in a form with extremely low metal (including, e.g., chromium) content.
Such compounds are conventionally prepared by methods of chemical synthesis which involve one or more oxidation steps which use chromium-based oxidizing agents. Consequently, the resulting product must undergo substantial purification in order to reduce the chromium content to acceptable levels.
Accordingly, there is a need for alternative methods of chemical synthesis of such thionine/thioninium compounds which avoid the need to use such metal-based (e.g., chromium-based) oxidizing agents.
The inventors have identified such methods, which are described herein. For example, thionine compounds of Formula (1) (such as LMT), thionine di-salt compounds of Formula (2) (such as LMT.2EsOH), and thioninium compounds of Formula (3) (such as MTC) can be prepared by methods which avoid the use of chromium oxidizing agents.
More specifically, the methods described herein include a step of preparing a substituted 10H-phenothiazine-3,7-diamine compound of Formula (1) by a step of selective alkylation by reductive amination, in which the corresponding unsubstituted diamine of Formula (4) is reacted with aldehyde/ketone, under reductive amination conditions.
Surprisingly and unexpectedly, the alkylation by reductive amination is selective, that is, the alkylation is selective for the pendant amino groups at the 3- and 7-positions in compounds of Formula (4), as compared to the bridging amino group at the 10-position in compounds of Formula (4). Surprisingly and unexpectedly, alkylation by reductive amination preferentially occurs at the pendant amino groups at the 3- and 7-positions, even to the point of di-alkylation at both of those positions, with little or no alkylation occurring at the bridging amino group at the 10-position.
Consequently (and surprisingly and unexpectedly), compounds of Formula (1) can be obtained in good yield without the use of chromium oxidizing agents, and thus without the need for further purification to remove residual chromium.